EP1075281A2 - Konjugate aus polyole und beta-interferon - Google Patents
Konjugate aus polyole und beta-interferonInfo
- Publication number
- EP1075281A2 EP1075281A2 EP99920094A EP99920094A EP1075281A2 EP 1075281 A2 EP1075281 A2 EP 1075281A2 EP 99920094 A EP99920094 A EP 99920094A EP 99920094 A EP99920094 A EP 99920094A EP 1075281 A2 EP1075281 A2 EP 1075281A2
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- EP
- European Patent Office
- Prior art keywords
- peg
- polyol
- ifn
- interferon
- conjugate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
Definitions
- the invention relates to polyol-IFN- ⁇ conjugates wherein a polyol unit is covalently bound to Cys 17 . Further objects of the present invention are the process for their site- specific production as well as their use in the therapy, prognosis or diagnosis of bacterial infections, viral infections, autoimmune diseases and inflammatory diseases.
- the present invention further relates to a method for the stepwise attachment of two or more PEG moieties to a polypeptide.
- Human fibroblast interferon has antiviral activity and can also stimulate natural killer cells against neoplastic cells. It is a polypeptide of about 20,000 Da induced by viruses and double-stranded RNAs . From the nucleotide sequence of the gene for fibroblast interferon, cloned by recombinant DNA technology, Derynk et al . (Nature, 285:542-547, 1980) deduced the complete amino acid sequence of the protein. It is 166 amino acid long. Shepard et al .
- -PEG- means "—CH 2 CH 2 0 (CH 2 CH 2 0) n CH 2 CH 2 - PEG is commonly used as methoxy-PEG-OH, (m-PEG) , in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to chemical modification.
- PEGs can be represented as R( -PEG-OH) m in which R represents a central core moiety such as pentaerythritol or glycerol, and m represents the number of branching arms.
- R represents a central core moiety such as pentaerythritol or glycerol
- m represents the number of branching arms.
- the number of branching arms (m) can range from three to a hundred or more.
- the hydroxyl groups are subject to chemical modification.
- Another branched form such as that described in PCT patent application WO 96/21469, has a single terminus that is subject to chemical modification.
- This type of PEG can be represented as (CH 3 0—PEG—) p R—X, whereby p equals 2 or 3 , R represents a central core such as lysine or glycerol, and X represents a functional group such as carboxyl that is subject to chemical activation.
- the "pendant PEG” has reactive groups, such as carboxyl, along the PEG backbone rather than at the end of PEG chains .
- the polymer can also be prepared with weak or degradable linkages in the backbone. For example, Harris has shown in U.S.
- Patent Application 06/026,716 that PEG can be prepared with ester linkages in the polymer backbone that are subject to hydrolysis. This hydrolysis results in cleavage of the polymer into fragments of lower molecular weight, according to the reaction scheme : -PEG-C0 2 -PEG- + H 2 0 - -PEG-C0 2 H + HO-PEG-
- the term polyethylene glycol or PEG is meant to comprise all the above described derivatives.
- the copolymers of ethylene oxide and propylene oxide are closely related to PEG in their chemistry, and they can be used instead of PEG in many of its applications. They have the following general formula:
- PEG is a useful polymer having the property of high water solubility as well as high solubility in many organic solvents. PEG is also non-toxic and non-immunogenic . When PEG is chemically attached (PEGylation) to a water insoluble compound, the resulting conjugate generally is water soluble, as well as soluble in many organic solvents.
- PEG-protein conjugates are currently being used in protein replacement therapies and for other therapeutic uses .
- PEGylated adenosine deaminase (ADAGEN ® ) is being used to treat severe combined immunodeficiency disease (SCIDS)
- PEGylated L-asparaginase (ONCAPSPAR ® ) is being used to treat acute lymphoblastic leukemia (ALL)
- PEGylated interferon- ⁇ is in Phase III trials for treating hepatitis C.
- PEG-protein conjugates with clinical efficacy see N.L. Burnham, Am. J. Hos . Pharm. , 15:210- 218, 1994.
- PEGylate proteins A variety of methods have been developed to PEGylate proteins. Attaching PEG to reactive groups found on the protein is typically done utilizing electrophilically activated PEG derivatives. Attaching PEG to the ⁇ - and e-amino groups found on lysine residues and the N-terminus results in a conjugate consisting of a mixture of products.
- conjugates consist of a population of the several PEG molecules attached per protein molecule ("PEGmers") ranging from zero to the number of amino groups in the protein.
- PEGmers For a protein molecule that has been singly modified, the PEG unit may be attached at a number of different amine sites. This type of non-specific PEGylation has resulted in a number of conjugates that become almost inactive. Reduction of activity is typically caused by shielding the protein's active binding domain as is the case with many cytokines and antibodies.
- Patent 4,917,888 describe the PEGylation of IFN- ⁇ and IL-2 with a large excess of methoxy-polyethylene glycolyl N- succinimidyl glutarate and methoxy-polyethylene glycolyl N- succinimidyl succinate. Both proteins were produced in microbial host cells, which allowed the site-specific mutation of the free cysteine to a serine. The mutation was necessary in microbial expression of IFN- ⁇ to facilitate protein folding.
- the IFN- ⁇ used in these experiments is the commercial product Betaseron ® , in which Cys 17 residue is replaced with a serine. Additionally, the absence of glycosylation reduced its solubility in aqueous solution. Non-specific PEGylation resulted in increased solubility, but a major problem was the reduced level of activity and yield.
- European Patent Application EP 593 868 entitled PEG- Interferon Conjugates, describes the preparation of PEG-IFN- conjugates. However, the PEGylation reaction is not site- specific, and therefore a mixture of positional isomers of PEG- IFN- ⁇ conjugates are obtained (see also Monkarsh et al . , ACS Symp. Ser. , 680:207-216, 1997). Kinstler et al . in European Patent Application EP 675
- cytokines as well as other proteins, do not possess a specific PEG attachment site and, apart from the examples mentioned above, it is very likely that some of the isomers produced through the PEGylation reaction be partially or totally inactive, thus causing a loss of activity of the final mixture .
- polyol-IFN- ⁇ conjugates and particularly PEG- IFN- ⁇ conjugates, are provided wherein a polyol unit is covalently bound to Cys 17 .
- the specific conjugation is obtained by allowing a thiol-reactive polyol agent to react with the Cys 17 residue in IFN- ⁇ .
- Such conjugates are expected to show increased effectiveness in vivo .
- the aim is to obtain increased solubility at neutral pH, increased stability (decreased aggregation) , decreased immunogenicity, and no loss of activity with respect to 'native' IFN- ⁇ .
- the results of such conjugation would decrease the number of doses for an intended effect, simplify and stabilize the formulation of ' a pharmaceutical composition, and possibly increase the long-term efficacy.
- the present invention further provides a method for the stepwise attachment of PEG moieties in series to a polypeptide.
- Figure 1 shows the Capillary Electrophoresis (CE) graph of the PEG-IFN- ⁇ conjugate prior to purification.
- Figures 2A-2C show the purification of the PEG-IFN- ⁇ conjugate carried out by size exclusion chromatography (Superose 12) : Fig. 2A - first pass; Fig. 2B - second pass; Fig. 2C - third pass.
- Figure 3 shows the SDS-PAGE chromatography of purified PEG-IFN- ⁇ conjugate from the third pass of chromatography.
- Lanes 1 and 4 are protein molecular weight standards
- lane 2 is "native" IFN- ⁇
- lane 3 is PEG-IFN- ⁇ conjugate.
- Figure 4 reports the Capillary Electrophoresis (CE) graph of purified PEG-IFN- ⁇ conjugate in which IFN- ⁇ is PEGylated with mPEG-OPSS 5k .
- Figure 5 reports the MALDI MS spectrum of purified
- Figure 6 shows a comparison between the anti-viral activity of "native" IFN- ⁇ and of PEG-IFN- ⁇ conjugate.
- WISH cells were incubated with indicated concentrations of IFN- ⁇ samples for 24 hours prior to challenge with cytopathic dose of vesicular stomatitis virus. The cytopathic effect was determined after an additional 48 hours by MTT conversion.
- Figure 7 shows the binding profile of IFN- ⁇ and PEG- IFN in Daudi cells.
- Figure 8 shows the pharmokinetic profile of IFN- ⁇ and
- Figure 9 shows the pharmokinetic profile of IFN- ⁇ and PEG-IFN in mice following subcutaneous administration.
- the dotted lines indicate assay LOQ for each standard curve.
- the present invention is based on the discovery that the attachment of a polyol moiety, more specifically a PEG moiety, to the Cys 17 residue of human IFN- ⁇ unexpectedly increased (or at least retained and did not result in a decrease) the IFN- ⁇ biological activity from that of native human interferon- ⁇ .
- a polyol moiety more specifically a PEG moiety
- the attachment of a polyol moiety, more specifically a PEG moiety to the Cys 17 residue of human IFN- ⁇ unexpectedly increased (or at least retained and did not result in a decrease) the IFN- ⁇ biological activity from that of native human interferon- ⁇ .
- this polyol-IFN- ⁇ conjugate also provides the desirable properties conferred by the polyol moiety, such as increased solubility.
- IFN- ⁇ means human fibroblast interferon, as obtained by isolation from biological fluids or as obtained by DNA recombinant techniques from prokaryotic or eukaryotic host cells as well as its salts, functional derivatives, precursors and active fractions, provided that they contain the cysteine residue appearing at position 17 in the naturally occurring form.
- the polyol moiety in the polyol- IFN- ⁇ conjugate according to the present invention can be any water-soluble mono- or bifunctional poly (alkylene oxide) having a linear or branched chain.
- the polyol is a poly (alkylene glycol) such as poly (ethylene glycol) (PEG) .
- PEG poly (ethylene glycol)
- other polyols such as, for example poly (propylene glycol) and copolymers of polyethylene glycol and polypropylene glycol, can be suitably used.
- PEG moiety is intended to include, but is not limited to, linear and branched PEG, methoxy PEG, hydrolytically or enzymatically degradable PEG, pendant PEG, dendrimer PEG, copolymers of PEG and one or more polyols, and copolymers of PEG and PLGA (poly (lactic/glycolic acid)) .
- salts refers both to salts of the carboxyl-groups and to the salts of the amino functions of the compound obtainable through known methods.
- the salts of the carboxyl-groups include inorganic salts as, for example, sodium, potassium, calcium salts and salts with organic bases as those formed with an amine as triethanolamine, arginine or lysine.
- the salts of the amino groups included for example, salts with inorganic acids as hydrochloric acid and with organic acids as acetic acid.
- the definition "functional derivatives" as herein used refers to derivatives which can be prepared from the functional groups present on the lateral chains of the amino acid moieties or on the terminal N- or C- groups according to known methods and are included in the present invention when they are pharmaceutically acceptable, i.e., when they do not destroy he protein activity or do not impart toxicity to the pharmaceutical compositions containing them.
- Such derivatives include for example esters or aliphatic amides of the carboxyl-groups and N-acyl derivatives of free amino groups or O-acyl derivatives of free hydroxyl-groups and are formed with acyl-groups as for example alcanoyl-- or aroyl-groups .
- the “precursors” are compounds which are converted into IFN- ⁇ in the human or animal body.
- active fractions of the protein refers to any fragment or precursor of the polypeptidic chain of the compound itself, alone or in combination with related molecules or residues bound to it, for example, residues of sugars or phosphates, or aggregates of the polypeptide molecule when such fragments or precursors show the same activity of IFN- ⁇ as medicament.
- conjugates of the present invention can be prepared by any of the methods known in the art . According to an embodiment of the invention, IFN- ⁇ is reacted with the
- PEGylating agent in a suitable solvent and the desired conjugate is isolated and purified, for example, by applying one or more chromatographic methods .
- Chromatographic method means any technique that is used to separate the components of a mixture by their application on a support (stationary phase) through which a solvent (mobile phase) flows.
- the separation principles of the chromatography are based on the different physical nature of stationary and mobile phase.
- Some particular types of chromatographic methods which are well-known in the literature, include: liquid, high pressure liquid, ion exchange, absorption, affinity, partition, hydrophobic, reversed phase, gel filtration, ultrafiltration or thin-layer chromatography.
- the "thiol-reactive PEGylating agent” as used in the present application, means any PEG derivative which is capable of reacting with the thiol group of the cysteine residue.
- the thiol-reactive PEGylating agent is the orthopyridyl disulfide (OPSS) derivative of PEG.
- OPSS orthopyridyl disulfide
- the PEGylating agent is used in its mono-methoxylated form where only one terminus is available for conjugation, or in a bifunctional form where both termini are available for conjugation, such- as for example in forming a conjugate with two IFN- ⁇ covalently attached to a single PEG moiety. It has preferably a molecular weight between 500 and 100,000.
- the second line of the above scheme reports a method for cleaving the PEG-protein linkage.
- the mPEG-OPSS derivative is highly selective for free sulphydryl groups and reacts rapidly under acidic pH conditions where the IFN- ⁇ is stable.
- the high selectivity can be demonstrated from the reduction of the conjugate to the native form of IFN- ⁇ and PEG.
- the disulfide bond that is produced between the protein and PEG moieties has been shown to be stable in the circulation, but it can be reduced upon entering the cell environment. Therefore it is expected that this conjugate, which does not enter the cell, will be stable in the circulation until it is cleared.
- the present invention is also directed to a method for the stepwise attachment of two or more PEG moieties to a polypeptide. This method is based upon the recognition that a low molecular weight activated PEG reacts more completely with a sterically hindered reaction site on a protein than does a high molecular weight activated PEG. PEG-modification of expensive therapeutic proteins must be cost effective in order for the production of the PEG conjugate to be practical.
- the conjugate in order to reduce glomerular filtration and optimize the pharmacological properties of the PEG-protein conjugate, the conjugate should have an effective size equivalent to that of a protein with a molecular weight of 70 kDa. This means that for a site specific modification where one PEG will be attached, a PEG derivative having a molecular weight of greater than 20 kDa is preferably attached. If the site of modification is sterically crowded, the reactive group on the large PEG moiety may have difficulty reaching the modification site and thus will lead to low yields.
- a preferred method of PEGylating a polypeptide according to the present invention increases the yield of site-specific PEGylation by first attaching a small hetero or homobifunctional PEG moiety that, due to its relatively smaller size, can react with sterically crowded sites. Subsequent attachment of a large molecular weight PEG derivative to the small PEG results in high yield of the desired PEGylated protein.
- the method for stepwise attachment of two or more PEG moieties in series to a polypeptide according to the present invention includes attaching a low molecular weight heterbifunctional or homobifunctional PEG moiety first to the polypeptide and then attaching a monofunctional or bifunctional PEG moiety to the free terminus of the low molecular weight PEG moiety that is attached to the polypeptide.
- the PEG-polypeptide conjugate can be purified using one or more of the purification techniques such as ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, and reverse phase chromatography.
- the low molecular weight PEG moiety has the formula:
- W and X are groups that independently react with an amine, sulfhydryl, carboxyl or hydroxyl functional group to attach the low molecular weight PEG moiety to the polypeptide.
- W and X are preferably independently selected from orthopyridyl disulfide, maleimides, vinyl sulfones, iodoacetamides, amines, thiols, carboxyls, active esters, benzotriazole carbonates, p- nitrophenol carbonates, isocyanates, and biotin.
- the low molecular weight PEG moiety preferably has a molecular weight in the range of about 100 to 5,000 daltons .
- the monofunctional or bifunctional PEG moiety for attachment to the free terminus of a low molecular weight PEG that is attached to the polypeptide has preferably a molecular weight in the range of about 100 daltons to 200 kDa and is preferably a methoxy PEG, branched PEG, hydrolytically or enzymatically degradable PEG, pendant PEG, or dendrimer PEG.
- the monofunctional or bifunctional PEG furthermore has the formula :
- Y is reactive to a terminal group on the free terminus of the low molecular weight PEG moiety that is attached to the polypeptide and Z is -OCH 3 or a group reactive with to form a bifunctional conjugate.
- the PEG-polypeptide conjugate produced by the above method for stepwise attachment of two or more PEG moieties can be used to produce a medicament or pharmaceutical composition for treating diseases or disorders for which the polypeptides is effective as an active ingredient.
- Another object of the present invention is to provide the conjugates in substantially purified form in order for them to be suitable for use in pharmaceutical compositions, as active ingredient for the treatment, diagnosis or prognosis of bacterial and viral infections as well as autoimmune, inflammatory diseases and tumors.
- Such pharmaceutical compositions represent a further object of the present invention.
- Non-limiting examples of the above-mentioned diseases include: septic shock, AIDS, rheumatoid arthritis, lupus erythematosus and- multiple sclerosis.
- An embodiment of the invention is the administration of a pharmacologically active amount of the conjugates of the invention to subjects at risk of developing one of the diseases reported above or to subjects already showing such pathologies.
- Any route of administration compatible with the active principle can be used.
- Parenteral administration such as subcutaneous, intramuscular or intravenous injection is preferred.
- the dose of the active ingredient to be administered depends on the basis of the medical prescriptions according to age, weight and the individual response of the patient.
- the dosage can be between 10 ⁇ g and 1 mg daily for an average body weight of 75 kg, and the preferable daily dose is between 20 ⁇ g and 200 ⁇ g.
- the pharmaceutical composition for parenteral administration can be prepared in an injectable form comprising the active principle and a suitable vehicle.
- Vehicles for the parenteral administration are well known in the art and include, for example, water, saline solution, Ringer solution and/or dextrose.
- the vehicle can contain small amounts of excipients in order to maintain the stability and isotonicity of the pharmaceutical preparation.
- the preparation of the solutions can be carried out according to the ordinary modalities.
- Recombinant human IFN- ⁇ stable at a concentration of 0.37 mg/ml in 50 M sodium acetate buffer, pH 3.6, was used for the preparation of a PEG-IFN- ⁇ conjugate.
- Approximately 1.0 ml of 6 M urea was added to 2 ml of IFN- ⁇ at a concentration of 0.37 mg/ml (0.74 mg, 3.7 x l "8 moles) .
- mPEG 5K -OPSS was added in a molar excess of 50 moles to one mole of IFN- ⁇ and the two were allowed to react in a polypropylene vial for either 2 hours at 37°C or 1 hour at 50°C.
- the reaction mixture was analyzed with Capillary Electrophoresis (CE) graph to determine the extent of PEG-IFN- ⁇ conjugate formation by the PEGylation reaction prior to any purification (Fig. 1) .
- a typical yield for this reaction is 50% PEG-IFN- ⁇ .
- the reaction products were filtered from the reaction mixture with a 0.22 mm syringe filter and the filtered solution was then loaded onto a size exclusion column (either Superose 12 or Superdex 75, Pharmacia) and eluted with 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 buffer.
- a size exclusion column either Superose 12 or Superdex 75, Pharmacia
- FIG. 2A shows the elution profile from the purification of the PEG-IFN- ⁇ conjugate on a Superose 12 size exclusion chromatography column.
- the peaks were collected and analyzed with SDS-PAGE (Fig. 3) .
- the fractions containing the PEG-IFN- ⁇ conjugate were pooled together and concentrate was then reloaded to the same size exclusion column to further purify the PEG-IFN- ⁇ conjugate due to the close proximity of the "native" IFN- ⁇ peak (Fig. 2B) . This procedure was repeated (third pass) to ensure purity (Fig. 2C) .
- Fig. 4 and Fig. 5 show the Capillary Electrophoresis graph and the MALDI MS spectrum, respectively, of the purified PEG- IFN- ⁇ conjugate.
- Recombinant human IFN- ⁇ was provided is stable in solution at 0.36 mg/ml in 50 mM sodium acetate buffer, pH 3.6. Approximately 36 mg of mPEG 30K -OPSS in 3 ml deionized H 2 0 was added to 3 ml of IFN- ⁇ at 0.36 mg/ml (1.08 mg, 4.9-xlO "8 moles) and the two were allowed to react in a polypropylene vial for 2 hours at 50°C. The reaction mixture was analyzed with capillary electrophoresis for extent of modification. Typical yields for this reaction are ⁇ 30%.
- the solution was then loaded onto a size exclusion column (Superose 12, Pharmacia) and eluted with 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 buffer. The peaks were collected and analyzed with SDS-PAGE for their contents.
- a size exclusion column Superose 12, Pharmacia
- Vesicular Stomatitis Virus stocks (ATCC V-520-001- 522) , stored at -70°C
- WISH Growth medium MEM high glucose with Earls salts + 10% FBS + 1.0% L-glutamine + Penicillin/Streptomycin (100 U/ml, 100 ⁇ g/ml)
- WISH Assay medium MEM high glucose with Earls salts + 5% FBS + 1.0% L-glutamine + Penicillin/Streptomycin (100 U/ml , 100 ⁇ g/ml)
- the plates are read at 595 nm using the Soft max Pro software package and Spectramax spectrophotometer system (Molecular Devices) .
- the PEG- IFN- ⁇ conjugate maintained a level of anti-viral activity superior to that of the freshly prepared parental lot of IFN- ⁇ .
- the observation that the PEG-IFN- ⁇ conjugate has approximately 4 -fold higher bioactivity than that of freshly prepared IFN- ⁇ may be also a consequence of the increased stability of the PEG- IFN- ⁇ conjugate with respect to the "native" IFN- ⁇ after addition of WISH cell assay medium.
- PEG[2 X 20 kD] -IFN- ⁇ was determined by WISH assay using the standard protocol described in Example 2 (Table 2) . Three independent assays were performed by three different individuals at separate times.
- the binding of PEG-IFN- ⁇ to its receptor on cells was evaluated in the presence of a fixed amount of 12S I-IFN- ⁇ 2a .
- IFN- ⁇ 2a was radiolabeled with 12S I using the chloramine T method.
- the 125 I bound IFN ⁇ 2a was removed from free iodine by running the reactants through a Sephadex G25 column and pooling the protein containing fractions (Pharmacia) .
- 12S I-IFN- ⁇ 2a was quantified by an IFN- ⁇ 2a ELISA assay (Biosource, USA) and the specific activity was determined.
- Daudi cells grown in the exponential phase of growth were harvested and 2 x 10 6 cells were incubated with 0.5 nM 12S I-IFN- ⁇ 2a for 3 hours at room temperature in the presence of different concentrations of PEG-IFN- ⁇ or IFN- ⁇ 2a diluted in an assay buffer which is RPMI 1640 containing 2% fetal bovine serum and 0.1% sodium azide.
- an assay buffer which is RPMI 1640 containing 2% fetal bovine serum and 0.1% sodium azide.
- the cells were spun through a layer of phthalate. oil and the cell bound radioactivity was counted on the gamma counter.
- the binding of PEG [30kD] -IFN- ⁇ and PEG [2 5 x 20kD] -IFN- ⁇ to the receptor were very similar or close to the binding activity of IFN- ⁇ as shown in Fig. 7.
- mice Twenty-eight female B6D2F1 strain mice (6-8 wks) (approximately 20g each) were separated into four groups as follows: Group 1 contained nine mice injected with a 200 ul single bolus of 500 ng/ml human IFN- ⁇ (final dose of 100 ng/ mouse) ; Group 2 (nine mice) received 200 ul of an equivalent mass of PEG30kD-IFN- ⁇ ; Group 3 received 200 ul of an equivalent mass of PEG(2 x 20 kD) -IFN- ⁇ ; and Group 4 is a group of three uninjected mice serving as a negative control.
- Blood samples (approximately 200 ul/sample) were collected at nine indicated times by disruption of the retro-orbital venous plexus with a capillary tube. Blood samples were allowed to clot for 1 hr at room temperature, rimmed and microcentrifuged. Sera removed therefrom were stored at -70°C until all samples were collected. Sera were assayed for the presence of bioactive human IFN- ⁇ using the Toray assay.
- Recombinant human interferon- ⁇ was provided in solution at 0.33 mg/ml in 50mM sodium acetate buffer, pH 3.8.. Approximately 3.6 mg (40 mole excess to moles of protein) of the heterobifunctional PEG reagent, OPSS-PEG 2k -hydrazide, in 2 ml deionized water was added to 3 ml of IFN- ⁇ at 0.33 mg/ml (0.99 mg) and the two were allowed to react in a polypropylene vial for 1 hour at 45°C. The reaction mixture was then analyzed with capillary electrophoresis to determine the extent of modification. Typical yields ranged from 90-97% that depended on the purity of the interferon ⁇ and PEG reagent.
- the solution was next loaded onto a size exclusion column (Superdex 75, Pharmacia) and eluted with 5 mM sodium phosphate, 150 mM NaCl, pH 7.0 buffer. The peaks were collected and analyzed with SDS- PAGE . The monoPEGylated interferon- ⁇ fractions were pooled together than used in a further modification step with high molecular weight PEG.
- Recombinant human interferon- ⁇ was provided in solution at 0.33 mg/ml in 50 mM sodium acetate buffer, pH 3.8. Approximately 6.1 mg (40 mole excess to moles of protein) of the homobifunctional PEG reagent, (OPSS) 2 -PEG 3400 , in 2 ml deionized water was added to 3 ml of interferon- ⁇ at 0.33 mg/ml (0.99 mg) and the two were allowed to react in a polypropylene vial for 2 hours at 50°C. The reaction was monitored with non-reducing SDS-PAGE and the final reaction mixture was analyzed with capillary electrophoresis to determine the extent of modification. Typical modifications for this reaction with interferon- ⁇ were >95%.
- the solution was then loaded onto a size exclusion column (Superdex 75, Pharmacia) and eluted with 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 buffer. The peaks were collected and analyzed with SDS-PAGE for their contents. The monoPEGylated interferon- ⁇ fractions were combined.
- Example 5 To the combined fractions of IFN-S-S-PEG 2k -Hydrazide in Example 5 was added mPEG 30k -ALD in a 20 mole excess to protein. The reaction was conducted at room temperature (25°C) for 4 hours and a sample was added to a size exclusion column (Superose 6, Pharmacia) to determine modification yield. The modification yield of this reaction was typically >80% depending upon the purity of the PEG reagent and reaction conditions.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK99920094T DK1075281T3 (da) | 1998-04-28 | 1999-04-28 | Polyol-IFN-beta-konjugater |
SI9930974T SI1421956T1 (sl) | 1998-04-28 | 1999-04-28 | Postopek za stopenjsko vezavo polietilenglikola (peg) na polipeptid |
SI9930662T SI1075281T1 (en) | 1998-04-28 | 1999-04-28 | Polyol-ifn-beta conjugates |
EP04003053A EP1421956B1 (de) | 1998-04-28 | 1999-04-28 | Verfahren zur schrittweise Bindung von Polyethylenglykol (PEG) an Polypeptide |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US8333998P | 1998-04-28 | 1998-04-28 | |
US83339P | 1998-04-28 | ||
PCT/US1999/009161 WO1999055377A2 (en) | 1998-04-28 | 1999-04-28 | Polyol-ifn-beta conjugates |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP04003053A Division EP1421956B1 (de) | 1998-04-28 | 1999-04-28 | Verfahren zur schrittweise Bindung von Polyethylenglykol (PEG) an Polypeptide |
Publications (2)
Publication Number | Publication Date |
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EP1075281A2 true EP1075281A2 (de) | 2001-02-14 |
EP1075281B1 EP1075281B1 (de) | 2004-09-08 |
Family
ID=22177687
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
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EP04003053A Expired - Lifetime EP1421956B1 (de) | 1998-04-28 | 1999-04-28 | Verfahren zur schrittweise Bindung von Polyethylenglykol (PEG) an Polypeptide |
EP99920094A Expired - Lifetime EP1075281B1 (de) | 1998-04-28 | 1999-04-28 | Konjugate aus polyole und beta-interferon |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
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EP04003053A Expired - Lifetime EP1421956B1 (de) | 1998-04-28 | 1999-04-28 | Verfahren zur schrittweise Bindung von Polyethylenglykol (PEG) an Polypeptide |
Country Status (31)
Country | Link |
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US (3) | US6638500B1 (de) |
EP (2) | EP1421956B1 (de) |
JP (2) | JP4574007B2 (de) |
KR (1) | KR100622796B1 (de) |
CN (2) | CN1187094C (de) |
AR (1) | AR020070A1 (de) |
AT (2) | ATE275422T1 (de) |
AU (1) | AU762621B2 (de) |
BG (2) | BG64694B1 (de) |
BR (1) | BR9910023A (de) |
CA (2) | CA2565375A1 (de) |
CY (1) | CY1108022T1 (de) |
CZ (2) | CZ298579B6 (de) |
DE (2) | DE69936409T2 (de) |
DK (2) | DK1421956T3 (de) |
EA (2) | EA003789B1 (de) |
EE (1) | EE05214B1 (de) |
ES (2) | ES2285286T3 (de) |
HK (2) | HK1038194A1 (de) |
HU (1) | HUP0300548A3 (de) |
IL (1) | IL139286A (de) |
NO (2) | NO329749B1 (de) |
NZ (1) | NZ507456A (de) |
PL (2) | PL196533B1 (de) |
PT (2) | PT1075281E (de) |
SI (2) | SI1075281T1 (de) |
SK (2) | SK286217B6 (de) |
TR (2) | TR200101751T2 (de) |
TW (2) | TWI266800B (de) |
UA (2) | UA79430C2 (de) |
WO (1) | WO1999055377A2 (de) |
Families Citing this family (78)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5932462A (en) | 1995-01-10 | 1999-08-03 | Shearwater Polymers, Inc. | Multiarmed, monofunctional, polymer for coupling to molecules and surfaces |
US7220717B2 (en) | 1997-08-14 | 2007-05-22 | Yeda Research And Development Company Ltd. | Interleukin-18 binding proteins, their preparation and use |
US7704944B2 (en) | 1997-08-14 | 2010-04-27 | Yeda Research And Development Company Ltd. | Interleukin-18 binding proteins, their preparation and use for the treatment of sepsis |
IL121860A0 (en) | 1997-08-14 | 1998-02-22 | Yeda Res & Dev | Interleukin-18 binding proteins their preparation and use |
PL200586B1 (pl) | 1998-10-16 | 2009-01-30 | Biogen Idec Inc | Polipeptydy zawierające mutanty interferonu-beta-1a, kodujące je cząsteczki kwasów nukleinowych, komórki gospodarza transformowane tymi cząsteczkami, sposób wytwarzania polipeptydów, zawierające je kompozycje farmaceutyczne i zastosowania polipeptydów |
AU762616B2 (en) * | 1998-10-16 | 2003-07-03 | Biogen Ma Inc. | Polymer conjugates of interferon beta-1a and uses |
US7238368B2 (en) * | 1999-04-23 | 2007-07-03 | Alza Corporation | Releasable linkage and compositions containing same |
MXPA01010750A (es) * | 1999-04-23 | 2003-08-20 | Alza Corp | Conjugado que tiene un enlace dividible para utilizarse en un liposoma. |
US7303760B2 (en) * | 1999-04-23 | 2007-12-04 | Alza Corporation | Method for treating multi-drug resistant tumors |
US7144574B2 (en) | 1999-08-27 | 2006-12-05 | Maxygen Aps | Interferon β variants and conjugates |
US7431921B2 (en) | 2000-04-14 | 2008-10-07 | Maxygen Aps | Interferon beta-like molecules |
US6531122B1 (en) | 1999-08-27 | 2003-03-11 | Maxygen Aps | Interferon-β variants and conjugates |
TR200101086A3 (de) * | 1999-10-15 | 2001-08-21 | ||
CA2395254C (en) | 1999-12-24 | 2010-05-11 | Kyowa Hakko Kogyo Co., Ltd. | Branched polyalkylene glycols |
CN1188172C (zh) | 2000-01-10 | 2005-02-09 | 马克西根控股公司 | G-csf偶联物 |
RU2278123C2 (ru) | 2000-02-11 | 2006-06-20 | Максиджен Холдингз Лтд. | Молекулы, подобные фактору vii или viia |
ATE471956T1 (de) | 2001-01-30 | 2010-07-15 | Kyowa Hakko Kirin Co Ltd | Verzweigte polyalkylenglykole |
EP1234583A1 (de) * | 2001-02-23 | 2002-08-28 | F. Hoffmann-La Roche Ag | PEG-Konjugate des HGF-NK4 |
MXPA03007619A (es) | 2001-02-27 | 2003-12-04 | Maxygen Aps | Nuevas moleculas similares a interferon beta. |
NZ530545A (en) | 2001-07-11 | 2006-10-27 | Maxygen Holdings Ltd | Specific conjugates comprising a polypeptide exhibiting G-CSF activity and a non-polypeptide moiety |
GEP20074024B (en) | 2002-01-18 | 2007-01-10 | Biogen Idec Inc | Polyalkylene glycol comprising a radical for conjugation of biologically active compound |
TWI334785B (en) | 2002-06-03 | 2010-12-21 | Serono Lab | Use of recombinant ifn-β1a and pharmaceutical composition comprising recombinant ifn-β1a for the treatment of hcv infection in patients of asian race |
WO2004020468A2 (en) * | 2002-08-28 | 2004-03-11 | Maxygen Aps | Interferon beta-like molecules for treatment of cancer |
US8129330B2 (en) * | 2002-09-30 | 2012-03-06 | Mountain View Pharmaceuticals, Inc. | Polymer conjugates with decreased antigenicity, methods of preparation and uses thereof |
WO2004060299A2 (en) * | 2002-12-26 | 2004-07-22 | Mountain View Pharmaceuticals, Inc. | Polymer conjugates of interferon-beta with enhanced biological potency |
RS20050501A (en) * | 2002-12-26 | 2007-08-03 | Mountain View Pharmaceuticals Inc., | Polymer conjugates of cytokines,chemokines,growth factors, polypeptide hormones and antagonists thereof with preserved receptor-binding activity |
ATE431403T1 (de) | 2003-03-20 | 2009-05-15 | Bayer Healthcare Llc | Fvii oder fviia varianten |
TWI272948B (en) | 2003-05-01 | 2007-02-11 | Ares Trading Sa | HSA-free stabilized interferon liquid formulations |
GB0316294D0 (en) * | 2003-07-11 | 2003-08-13 | Polytherics Ltd | Conjugated biological molecules and their preparation |
ES2428358T3 (es) | 2003-10-17 | 2013-11-07 | Novo Nordisk A/S | Terapia de combinación |
AU2008201682B2 (en) * | 2004-02-02 | 2011-02-24 | Ambrx, Inc. | Modified human interferon polypeptides and their uses |
AU2005211385B2 (en) * | 2004-02-02 | 2008-12-11 | Ambrx, Inc. | Modified human growth hormone polypeptides and their uses |
EP1586334A1 (de) * | 2004-04-15 | 2005-10-19 | TRASTEC scpa | G-CSF Konjugate mit PEG |
US7879320B2 (en) | 2004-05-17 | 2011-02-01 | Ares Trading S.A. | Hydrogel interferon formulations |
EA010979B1 (ru) | 2004-06-01 | 2008-12-30 | Арес Трейдинг С.А. | Стабилизированные жидкие препаративные формы интерферона |
WO2005117948A1 (en) | 2004-06-01 | 2005-12-15 | Ares Trading S.A. | Method of stabilizing proteins |
TW200633718A (en) * | 2004-12-16 | 2006-10-01 | Applied Research Systems | Treatment of hepatitis c in the asian population |
ATE494012T1 (de) * | 2004-12-21 | 2011-01-15 | Nektar Therapeutics | Stabilisierte polymer-thiol-reagenzien |
US7816320B2 (en) | 2004-12-22 | 2010-10-19 | Ambrx, Inc. | Formulations of human growth hormone comprising a non-naturally encoded amino acid at position 35 |
ATE542920T1 (de) | 2004-12-22 | 2012-02-15 | Ambrx Inc | Modifiziertes menschliches wachstumshormon |
MX2007007591A (es) | 2004-12-22 | 2007-07-25 | Ambrx Inc | Metodos para expresion y purificacion de hormona de crecimiento humano recombinante. |
WO2006134173A2 (en) | 2005-06-17 | 2006-12-21 | Novo Nordisk Health Care Ag | Selective reduction and derivatization of engineered proteins comprising at least one non-native cysteine |
EP2099495A2 (de) * | 2005-08-04 | 2009-09-16 | Nektar Therapeutics AL, Corporation | Konjugate eines g-csf-teils und eines polymers |
EP1917276B1 (de) | 2005-08-26 | 2018-03-21 | Ares Trading S.A. | Verfahren zur herstellung von glykosyliertem interferon-beta |
CA2616479A1 (en) | 2005-09-01 | 2007-03-08 | Ares Trading S.A. | Treatment of optic neuritis |
US20070238656A1 (en) * | 2006-04-10 | 2007-10-11 | Eastman Kodak Company | Functionalized poly(ethylene glycol) |
US20080096819A1 (en) * | 2006-05-02 | 2008-04-24 | Allozyne, Inc. | Amino acid substituted molecules |
EP2018437A2 (de) * | 2006-05-02 | 2009-01-28 | Allozyne, Inc. | Mit nicht-natürlichen aminosäuren substituierte polypeptide |
CN104593348A (zh) | 2006-05-24 | 2015-05-06 | 诺沃—诺迪斯克保健股份有限公司 | 具有延长的体内半寿期的因子ix类似物 |
WO2007135172A2 (en) | 2006-05-24 | 2007-11-29 | Laboratoires Serono S.A. | Cladribine regimen for treating multiple sclerosis |
CA2685596A1 (en) | 2007-05-02 | 2008-11-13 | Ambrx, Inc. | Modified interferon beta polypeptides and their uses |
CA2707840A1 (en) | 2007-08-20 | 2009-02-26 | Allozyne, Inc. | Amino acid substituted molecules |
WO2009080699A2 (en) * | 2007-12-20 | 2009-07-02 | Merck Serono S.A. | Peg-interferon-beta formulations |
JP5702150B2 (ja) | 2008-02-08 | 2015-04-15 | アンブルックス, インコーポレイテッドAmbrx, Inc. | 修飾されているレプチンポリペプチドおよびそれらの使用 |
CN101525381B (zh) * | 2008-03-04 | 2012-04-18 | 北京百川飞虹生物科技有限公司 | 一种重组复合干扰素及其表达载体的构建和表达 |
US20100112660A1 (en) * | 2008-05-30 | 2010-05-06 | Barofold, Inc. | Method for Derivatization of Proteins Using Hydrostatic Pressure |
US20110124614A1 (en) * | 2008-10-25 | 2011-05-26 | Halina Offner | Methods And Compositions For The Treatment of Autoimmune Disorders |
DE102009032179A1 (de) * | 2009-07-07 | 2011-01-13 | Biogenerix Ag | Verfahren zur Reinigung von Interferon beta |
ES2365343B1 (es) | 2009-11-19 | 2012-07-10 | Fundación Centro Nacional De Investigaciones Cardiovasculares Carlos Iii | Uso de cd98 como marcador de receptividad endometrial. |
JP5914363B2 (ja) | 2010-02-16 | 2016-05-11 | ノヴォ ノルディスク アー/エス | 低減されたvwf結合を有する因子viii分子 |
AR080993A1 (es) | 2010-04-02 | 2012-05-30 | Hanmi Holdings Co Ltd | Formulacion de accion prolongada de interferon beta donde se usa un fragmento de inmunoglobulina |
US20120135912A1 (en) | 2010-05-10 | 2012-05-31 | Perseid Therapeutics Llc | Polypeptide inhibitors of vla4 |
EP2593130A2 (de) | 2010-07-15 | 2013-05-22 | Novo Nordisk A/S | Stabilisierte faktor-viii-varianten |
JP6042335B2 (ja) | 2010-09-15 | 2016-12-14 | ノヴォ ノルディスク アー/エス | 細胞取込みが低下した第viii因子変異体 |
US9913880B2 (en) | 2011-02-18 | 2018-03-13 | Stemdr Inc. | Method of treating sepsis or septic shock |
MX2014000031A (es) | 2011-07-01 | 2014-07-09 | Bayer Ip Gmbh | Polipeptidos de fusion de relaxina y usos de los mismos. |
US10358470B2 (en) * | 2011-10-01 | 2019-07-23 | Glytech, Inc. | Glycosylated polypeptide and pharmaceutical composition containing same |
CN117462693A (zh) | 2012-02-27 | 2024-01-30 | 阿穆尼克斯运营公司 | Xten缀合组合物和制造其的方法 |
CN104136038A (zh) | 2012-02-29 | 2014-11-05 | 东丽株式会社 | 体腔积液抑制剂 |
MY171183A (en) | 2013-03-29 | 2019-09-30 | Glytech Inc | Polypeptide glycosylated with sialylated sugar chain |
KR101671501B1 (ko) * | 2014-07-24 | 2016-11-03 | 에이비온 주식회사 | 페길화된 인터페론-베타 변이체 |
WO2016013697A1 (ko) * | 2014-07-24 | 2016-01-28 | 에이비온 주식회사 | 인터페론-베타 변이체의 폴리에틸렌글리콜 배합체 |
SI3183264T1 (sl) | 2014-08-19 | 2021-03-31 | Biogen Ma Inc. | Metoda pegilacije |
CN115919996A (zh) | 2015-05-01 | 2023-04-07 | 雅利斯塔制药公司 | 用于治疗眼科疾病的脂联素拟肽 |
PE20180498A1 (es) * | 2015-06-19 | 2018-03-09 | Eisai Randd Man Co Ltd | Inmunoglobulinas conjugadas en cys80 |
WO2018195006A1 (en) * | 2017-04-17 | 2018-10-25 | The Regents Of The University Of Colorado, A Body Corporate | Optimization of enzyme replacement therapy for treatment of homocystinuria |
US20220220176A1 (en) | 2019-01-28 | 2022-07-14 | Toray Industries, Inc. | Polyethylene glycol-modified form of hepatocyte growth factor or active fragment thereof |
US20220118050A1 (en) | 2019-01-28 | 2022-04-21 | Toray Industries, Inc. | Polyethylene glycol-modified form of hepatocyte growth factor or active fragment thereof |
Family Cites Families (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4766106A (en) | 1985-06-26 | 1988-08-23 | Cetus Corporation | Solubilization of proteins for pharmaceutical compositions using polymer conjugation |
EP0229108B1 (de) * | 1985-06-26 | 1990-12-27 | Cetus Corporation | Solubilisierung von proteinen für pharmazeutische zusammensetzungen mittels polymerkonjugierung |
US4917888A (en) | 1985-06-26 | 1990-04-17 | Cetus Corporation | Solubilization of immunotoxins for pharmaceutical compositions using polymer conjugation |
US5206344A (en) * | 1985-06-26 | 1993-04-27 | Cetus Oncology Corporation | Interleukin-2 muteins and polymer conjugation thereof |
US5208344A (en) * | 1987-07-31 | 1993-05-04 | American Home Products Corporation | Naphthalenepropionic acid derivatives as anti-inflammatory/antiallergic agents |
US5166322A (en) * | 1989-04-21 | 1992-11-24 | Genetics Institute | Cysteine added variants of interleukin-3 and chemical modifications thereof |
US5595732A (en) * | 1991-03-25 | 1997-01-21 | Hoffmann-La Roche Inc. | Polyethylene-protein conjugates |
US5382657A (en) * | 1992-08-26 | 1995-01-17 | Hoffmann-La Roche Inc. | Peg-interferon conjugates |
DK0730470T3 (da) | 1993-11-10 | 2002-06-03 | Enzon Inc | Forbedrede interferonpolymerkonjugater |
RO115788B1 (ro) * | 1994-03-31 | 2000-06-30 | Amgen Inc. | Polipeptidă mgdf, derivat de polipeptidă mgdf, polipeptidă mgdf mono-pegilată şi procedee de obţinere a acestora |
AU696387B2 (en) * | 1994-05-18 | 1998-09-10 | Inhale Therapeutic Systems, Inc. | Methods and compositions for the dry powder formulation of interferons |
US5824784A (en) * | 1994-10-12 | 1998-10-20 | Amgen Inc. | N-terminally chemically modified protein compositions and methods |
US5932462A (en) | 1995-01-10 | 1999-08-03 | Shearwater Polymers, Inc. | Multiarmed, monofunctional, polymer for coupling to molecules and surfaces |
SE9503380D0 (sv) * | 1995-09-29 | 1995-09-29 | Pharmacia Ab | Protein derivatives |
TW517067B (en) * | 1996-05-31 | 2003-01-11 | Hoffmann La Roche | Interferon conjugates |
US5851984A (en) * | 1996-08-16 | 1998-12-22 | Genentech, Inc. | Method of enhancing proliferation or differentiation of hematopoietic stem cells using Wnt polypeptides |
KR0176625B1 (ko) | 1996-11-05 | 1999-04-01 | 삼성전자주식회사 | 적외선 물체검출장치 |
JP2001508783A (ja) * | 1997-01-29 | 2001-07-03 | ポリマスク・ファーマシューティカルズ・パブリック・リミテッド・カンパニー | Peg化法 |
EP1881005B1 (de) * | 1997-07-14 | 2013-04-03 | Bolder Biotechnology, Inc. | Derivate von G-CSF und damit zusammenhängende Proteine |
US6307024B1 (en) * | 1999-03-09 | 2001-10-23 | Zymogenetics, Inc. | Cytokine zalpha11 Ligand |
US6531122B1 (en) * | 1999-08-27 | 2003-03-11 | Maxygen Aps | Interferon-β variants and conjugates |
-
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2001
- 2001-12-24 HK HK01109037A patent/HK1038194A1/xx not_active IP Right Cessation
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2003
- 2003-08-28 US US10/649,609 patent/US7357925B2/en not_active Expired - Fee Related
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2005
- 2005-11-17 HK HK05110273A patent/HK1076115A1/xx not_active IP Right Cessation
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2007
- 2007-02-13 US US11/674,476 patent/US7700314B2/en not_active Expired - Fee Related
- 2007-07-19 CY CY20071100966T patent/CY1108022T1/el unknown
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2010
- 2010-03-09 NO NO20100324A patent/NO332224B1/no not_active IP Right Cessation
- 2010-04-26 JP JP2010100840A patent/JP2010184929A/ja active Pending
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